Human cells contain a proteinaceous structural framework called a cytoskeleton. Each cell type contains a different protein or proteins as its cytoskeleton. For example, a brain cell's cytoskeleton is different than a muscle cell's or a lung cell's. In short, one means of differentiating cells is through an analysis of the proteins of the cytoskeleton.
Such differentiation, however, was only a theoretical concept due to impossibility of differentiating epithelial cells. Epithelial cells form the epidermis and line hollow organs and all passages of the respiratory, digestive, and genito-urinary systems. These are the host common cells of the human body. Therein, however, lies the problem. It was discovered that epithelial cells could be differentiated from the other major cell types (mesenchymal, glial, neuronal, and muscle cells) by the presence of keratin proteins in the cytoskeleton.
The intermediate filaments found in keratinocytes and other epithelial cells are composed of about 20 different keratin subunits. Each of these 20 proteins differ by as little as 10-12 amino acid sequences. The subunits expressed in a given epithelial cell vary widely depending on cell type, period of embryonic development, degree of differentiation, and growth environment of the cell. Changes in the pattern of keratin synthesis have also been observed during experimental skin carcinogenesis in established cell lines of malignant keratinocytes and in a variety of pathologic processes in the skin. Development of antisera specific to any one keratin subunit is, therefore, very useful for studies concerning expression and function of individual keratin proteins. This has proved difficult, however, since both polyclonal antibodies prepared against purified keratin subunits and monoclonal antibodies recognize more than one subunit. This shows that these antibodies are elicited against structural features common among keratin subunits. It is this similarity between the various keratin proteins that has made differentiation between epithelial cells most difficult.
The first tests developed to differentiate between epithelial cells involve monoclonal antibodies. These antibodies differentiate epithelial cells from non-epithelial cells, but they exhibit only limited utility in differentiating between epithelial cells. Monoclonal antibodies have been developed that are specific for groups of keratin proteins.
The difficulty in developing highly specific monoclonal antibodies or a differentiating reagent lies in the inability to determine the exact amino acid sequence of the protein. Antibodies developed prior to this invention are specific for amino acid sequences of the keratin protein common to all of the keratins and are, therefore, not individually specific.
The present invention discloses a method for producing monospecific antibodies to the individual keratin proteins. The reagents of this invention, therefore, are useful as diagnostic and differentiation reagents. More importantly, perhaps, these antibodies are useful in detecting and identifying specific types of carcinomas, mesotheliomas, adenocarcinomas, and other forms of malignancies arising from keratin-containing cells. The antibodies of the invention are, therefore, useful in identifying the site of origin of malignancies.
The present invention was made possible by the discovery of two technical advances of the invention: (1) cloning the gene for the keratin protein; and (2) establishing the amino acid sequences from the cloned keratin gene. The combination of these discoveries led to the elucidation of the amino acid sequences for each keratin protein. In broad terms, the invention is the development of antibodies which specifically react with sequences peculiar to each keratin protein. Exemplified are the methods for producing antibodies specific for keratins of 55 kD, 59 kD, 60 kD, and 67 kD molecular weight.